Moraxella (Branhamella) catarrhalis is a pathogenic bacterium, recognized as the third most common causative agent of otitis media and sinusitis in children, after Streptococcus pneumoniae and Haemophilus influenzae (Bluestone, C. D., 1986, Drugs 31(Suppl 3):132-41; Catlin, B. W., 1990, Clin. Microbiol. Rev. 3:293-320; Doem, G. V., 1986, Diagn. Microbiol. Infect. Dis. 4:191-201; Enright, M. C. and H. McKenzie, 1997, J. Med. Microbiol. 46:360-71; Faden, H., et al., 1994, J. Infect. Dis. 169:1312-1317). This Gram-negative diplococcus also causes respiratory tract infections in adults (Boyle, F. M., et al., 1991, Med. J. Aust. 154:592-596; Sarubbi, F. A., et al., 1990, Am. J. Med. 88:9S-14S), especially those who are immunocompromised or have chronic obstructive pulmonary diseases (Enright, M. C. and H. McKenzie, 1997, J. Med. Microbiol. 46:360-371). The incidence of disease caused by M. catarrhalis appears to be increasing (McLeod, D. T., et al., 1986, Br. Med. J. 292:1103-1105; Fung, C.P., et al., 1992, J. Antimicrob. Chemother. 30:47-55). Currently, there is no vaccine for M. catarrhalis-mediated diseases.
Although protective antigens of M. catarrhalis have not been clearly defined, development of serum antibodies against M. catarrhalis appears to be important in immunity against M. catarrhalis. For example, normal adults with immunity resulting from natural colonization or infection have a lower carriage rate (1 to 6%) than children (50 to 78%) and elderly persons ( greater than 26%), and suffer fewer infections (Ejlertsen T. et al., 1994, J. Infect. 29:23-31; Faden H. et al., 1994, J. Infect. Dis. 169:1312-1317; Eliasson, I., 1986, Drugs 31 (Suppl 3):7-10; Vaneechoutte, M., et al., 1990., J. Clin. Microbiol. 28:2674-2680). Children develop serum antibodies to M. catarrhalis gradually during the first four years of life, which seems to correlate with a decrease in the incidence of bacteremia and otitis media caused by M. catarrhalis (CDR Weekly Reports, 1992-1995, Communicable Disease Surveillance Centre, London; Goldblatt D., et al., 1990, J. Infect. Dis. 162:1128-1135; Vaneechoutte, M., et al., 1990., J Clin Microbiol 28:2674-2680; Bluestone, C. D., 1986, Drugs 31 (Suppl 3):132-141). Antibodies to M. catarrhalis have also been detected in acute and in convalescent sera of adult patients (Christensen, J. J., et al., 1990, Clin. Diagn. Lab. Immunol. 3:717-721; Rahman, M., et al., 1997, APMIS 105:213-220). Most convalescent sera demonstrate bactericidal activity against the corresponding M. catarrhalis isolate (Chapman, A. J. Jr., et al., 1985, J. Infect. Dis. 151:878-882). These results indicate that serum antibodies are likely to =be involved in protection against infections with M. catarrhalis. 
Efforts to date to study M. catarrhalis as an important pathogen have generally focused on describing surface antigens, such as outer membrane proteins (OMP) (Bhushan, R., et al., 1994, J. Bacteriol. 176:6636-6643; Campagnari, A. A., et al., 1994, Infect. Immun. 62:4909-4914; Helminen, M. E., et al., 1993, Infect. Immun. 61:2003-2010; Helminen, M. E., et al., 1994, J. Infect. Dis. 170:867-872; Murphy, T. F., et al., 1993, Mol. Microbiol. 10:87-97). Two outer membrane proteins that have been extensively studied are a high-molecular-weight protein (UspA) and a major outer membrane protein (CD). Both of these proteins are relatively conserved among different strains of M. catarrhalis and are able to generate bactericidal antibodies (Helminen, M. E., et al., 1994, J. Infect. Dis. 170:867-872; Murphy, T. F., et al., 1993, Mol. Microbiol. 10:87-97; Yang, Y. P., et al., 1997, FEMS Immunol. Med. Microbiol. 17:187-199). In addition, passive immunization with monoclonal antibodies to UspA, or immunization with UspA, has resulted in enhanced pulmonary clearance of M. catarrhalis strains in a murine model (Helminen, M. E., et al., 1994, J. Infect. Dis. 170:867-872; Chen, D., et al., 1996, Infect. Immun. 64:1900-1905). Genes encoding CD protein have been cloned and sequenced (Murphy et al., 1993, Molec. Microbiol. 10(1):87).
Other outer membrane proteins that have been purified and characterized include protein E (OMP E) (Bhushan et al., 1994, J. Bacteriol., 176(21):6636), protein B1 (Ducey et al., 1996, Abstracts, Gen. Mtg. Am. Soc. Microbiol., 96(0):186), and protein COPB (Aebi et al., 1996, Abstracts, Intersci. Conf. Antimicrobial Agents and Chemotherapy 36:158). Other surface antigens include fimbriae, which have not been found in all isolates (Marrs, C. F. and S. Weir, 1990, Am. J. Med. 88 (suppl 5A):36S-40S), and a capsular polysaccharide, whose existence is controversial (Ahmed, K., et al., 1991, Microbiol. Immunol. 35:361-366). Lipooligosaccharide-associated high molecular weight outer membrane protein has also been identified (Klingman and Murphy, 1992, Abstr. Gen Mtg. Am. Soc. Microbiol.).
Lipooligosaccharide (LOS), a major surface component of M. catarrhalis, is a virulence factor for the pathogenesis of the bacterial infections (Doyle, W. J., 1989, Pediatr. Infect. Dis. J. 81(Suppl): S45-S47; Fomsgaard, J. S., et al., 1991, Infect. Immun. 59:3346-3349). The LOS may be important for development of immunoprotection because (1) serum antibodies to LOS have been detected in patients with M. catarrhalis infections, (2) the convalescent-phase IgG anti-LOS from patients has demonstrated bactericidal activity against M. catarrhalis strains, and (3) LOS appears to have a conserved structure based on its serological properties in humans (Rahman, M., et al., 1995, Eur. J. Clin. Microbiol. Infect. Dis. 14:297-304; Tanaka, H., et al., 1992, J. Jpn. Assoc. Infect. Dis. 66: 709-715). Similarly, serum bactericidal LPS or PS antibodies specific to other microorganisms (e.g., H. influenzae type b, Neisseria meningitidis, Vibrio cholerae, Shigella sonnei) confer immunity to those pathogens in humans (Robbins, J.B., et al., 1995, J. Infect. Dis. 171:1387-1398; Cohen, D., et al., 1997, Lancet 349:155-159).
Three major antigenic types (A, B and C) of LOS account for about 95% of M. catarrhalis strains (i.e., 61% A; 29% B; and 5% C in one study) (Vaneechoutte, M., et al., 1990, J. Clin. Microbiol. 28:182-187). Studies have shown that these LOSs contain an oligosaccharide linked to lipid A, without an 0-specific polysaccharide, and the oligosaccharides from the three serotypes are branched with a common inner core (Edebrink, P., et al., 1994, Carbohydr. Res. 257:269-284; Edebrink, P., et al., 1995, Carbohydr. Res. 266:237-261; Edebrink, P., et al., 1996, Carbohydr. Res. 295:127-146).
Lipopolysaccharide (LPS) and LOS from a variety of microorganisms are generally toxic in vivo to mammals. Many approaches have been used to detoxify LPS or LOS, or to obtain nontoxic polysaccharides from LPS or oligosaccharides from LOS. For example, mild-acid treatment of LPS or LOS has been used to cleave the lipid A portion from the LOS molecule at the Kdo-glucosamine linkage (Gu, X. X., and C. M. Tsai, 1993, Infect. Immun. 61:1873-1880). Another method is mild-alkali treatment of LOS, removes ester-linked fatty acids while preserving amide-linked fatty acids of lipid A (Gupta, R. K., et al., 1992, Infect. Immun. 60:3201-3208; Gu et al., 1996, Infect. and Imm. 64(10):4047).
Development of vaccines against M. catarrhalis and other microorganisms has been attempted using a variety of approaches (Karma et al., 1995, Intl. J. Ped. Otorhinolaryngol. 32 (SUPPL.): S127-S134). Vaccines against M. catarrhalis based on outer membrane proteins E and CD, derived peptides and oligopeptides, or nucleotides encoding these proteins have been disclosed in U.S. Pat. No. 5,607,846 and U.S. Pat. No. 5,556,755. Conjugate vaccines made up of a carbohydrate-containing antigen bound to an immunomodulating cytokine, lymphokine, hormone or growth factor have been disclosed in U.S. Pat. No. 5,334,379. Canadian Pat. No. 2,162,193 discloses that a purified bacterial lactoferrin receptor protein may be used as a vaccine against pathogens that produce a lactoferrin receptor protein, including M. caiarrhalis. PCT Application WO 90/11777 discloses a method for obtaining unassembled bacterial pilus subunits for use in a vaccine against M. catarrhalis and other bacteria.
A vaccine against M. catarrhalis that is both nontoxic and immunogenic is needed to prevent otitis media, sinusitis and similar respiratory tract infections in mammals, particularly in human children and adults. Although methods of detoxification of LOS from other microorganisms are known, the detoxified products (i.e., hapten) are generally poorly immunogenic in vivo. Therefore, there is a need for a form of M. catarrhalis LOS that is detoxified but sufficiently immunogenic to elicit an immune response with production of anti-LOS antibodies, preferably IgG, in vivo in mammals.
According to one aspect of the invention, there is disclosed conjugate vaccine for Moraxella catarrhalis, including a lipooligosaccharide (LOS) isolated from M. catarrhalis and detoxified by treating to remove esterified fatty acids to produce a detoxified LOS (dLOS), or by treating to remove lipid A to produce an oligosaccharide (OS), and an immunogenic carrier covalently linked thereto. In one embodiment, the immunogenic carrier is a protein. In another embodiment, the immunogenic carrier protein is selected from the group consisting of UspA isolated from M. calarrhalis, CD isolated from M. catarrhalis, tetanus toxin/toxoid, a high molecular weight protein (HMP) isolated from nontypeable Haemophilus influenzae, diphtheria toxin/toxoid, detoxified P. aeruginosa toxin A, cholera toxin/toxoid, pertussis toxin/toxoid, Clostridium perfringens exotoxins/toxoid, hepatitis B surface antigen, hepatitis B core antigen, rotavirus VP 7 protein; CRMs (Cross Reacting Materials), including CPM197 (Pappenheimer et al., Immunochem. 9:891-906, 1972) and CRM3201 (Black et al., Science 240:656-659, 1988); and respiratory syncytial virus F and G protein. In one aspect of the vaccine, the immunogenic carrier protein is tetanus toxoid or HMP. Another embodiment is a pharmaceutical composition that includes such a vaccine conjugate in a pharmaceutically acceptable carrier, which may include an adjuvant. Preferably, the adjuvant is an admixture of monophosphoryl lipid A and trehalose dimycolate or alum. In one embodiment, the immunogenic carrier is covalently linked to de-esterified LOS via a linker compound. Preferably, the linker compound is selected from the group consisting of adipic acid dihydrazide, E-aminohexanoic acid, chlorohexanol dimethyl acetal, D-glucuronolactone and p-nitrophenylethyl amine, and more preferably, the linker compound is adipic acid dihydrazide. In one embodiment, the vaccine further includes an oligosaccharide (OS) isolated from M. catarrhalis by removal of lipid A from LOS, which is covalently linked to an immunogenic carrier.
According to another aspect of the invention, there is disclosed a lipooligosaccharide isolated from Moraxella catarrhalis and detoxified by removal of ester-linked fatty acids therefrom (dLOS), or an oligosaccharide obtained from removal of lipid A from LOS. In one embodiment, the Moraxella catarrhalis from which the lipooligosaccharide is isolated is a purified strain of Moraxella catarrhalis. 
According to another aspect of the invention, there is disclosed a method of preventing otitis media caused by infection with Moraxella catarrhalis in a manmmal, including administering to the manmmal an effective immunoprotective amount of the conjugate vaccine that includes a detoxified lipooligosaccharide (dLOS) produced by de-esterification of LOS derived from Moraxella catarrhalis, or an oligosaccharide (OS) produced by removal of lipid A from LOS, and an immunogenic carrier covalently linked to the dLOS or to the OS. In a preferred embodiment, the mammal is a human. In another embodiment, the conjugate vaccine is administered parenterally. In one embodiment, the conjugate vaccine is administered by intramuscular injection, subcutaneous injection, or by deposit on intranasal mucosal membrane or combinations thereof. In another embodiment, the effective immunoprotective amount is between about 10 xcexcg and about 50 xcexcg per dose. The method may also include injecting between about 10 xcexcg and about 25 xcexcg of the conjugate vaccine at about two months and again at about thirteen months after the administering step. In one embodiment, the administering step includes administering a first dose, and then administering a second dose of about 10 xcexcg to about 25 xcexcg of the conjugate vaccine at about two months after the first dose, administering a third dose of about 10 xcexcg to about 25 xcexcg of the conjugate vaccine at about 2 months after the second dose, and administering a fourth dose of about 10 xcexcg to about 25 xcexcg of the conjugate vaccine at about 12 months after the third dose.
According to another aspect of the invention, there is disclosed a method for detoxifying lipooligosaccharide (LOS) isolated from Moraxella catarrhalis, including removing ester-linked fatty acids from the LOS. In one embodiment, the ester-linked fatty acids are removed with hydrazine or a mild alkaline reagent.
The invention also includes a method for detoxifying LOS from Moraxella catarrhalis, including removal of lipid A from the LOS to produce OS. In one embodiment, the lipid A is removed by acid treatment.
According to another aspect of the invention, there is disclosed a method of making a conjugate vaccine against Moraxella catarrhalis including removing ester-linked fatty acids from lipooligosaccharide (LOS) isolated from M. catarrhalis to produce de-esterified LOS (dLOS); and covalently linking the dLOS to an immunogenic carrier.
In one embodiment, the removing step comprises treating the LOS with hydrazine or a mild alkaline reagent. In one embodiment, the linking step includes attaching the dLOS to a linker compound and attaching the linker compound to the immunogenic carrier.
Preferably, the linker compound is adipic acid dihydrazide, xcex5-aminohexanoic acid, chlorohexanol dimethyl acetal, D-glucuronolactone or p-nitrophenylethyl amine, and more preferably, the linker compound is adipic acid dihydrazide. In another embodiment, the vaccine composition may include an adjuvant.
The present invention also provides a conjugate vaccine comprising a lipooligosaccharide (LOS) isolated from M. catarrhalis and detoxified by treating to remove esterified fatty acids to produce detoxified LOS (dLOS), or by removing lipid A to produce oligosaccharide (OS), and an immunogenic carrier covalently linked thereto, for use in preventing otitis media caused by infection with Moraxella catarrhalis in a mammal. Preferably, the immunogenic carrier is a protein. In one aspect of this preferred embodiment, immunogenic carrier protein is UspA isolated from M. catarrhalis, CD isolated from M. catarrhalis, tetanus toxin/toxoid, a high molecular weight protein (HMP) isolated from nontypeable Haemophilus influenzae, diphtheria toxin/toxoid, detoxified P. aeruginosa toxin A, cholera toxin/toxoid, pertussis toxin/toxoid, Clostridium perfringens exotoxins/toxoid, hepatitis B surface antigen, hepatitis B core antigen, rotavirus VP 7 protein; CRMs including CRM197 (Pappenheimer et al. supra.) and CRM3201, (Black et al., supra.); or respiratory syncytial virus F and G protein. Preferably, the immunogenic carrier protein is tetanus toxoid or HMP.